Early metabolic and hemodynamic indicators of kidney dysfunction in mice offspring from parental low protein diet.

BACKGROUND: Parental malnutrition, particularly a low-protein diet (LPD), causes oligonephropathy at birth and predisposes offspring to hypertension and chronic kidney disease later in life. The onset of adult kidney disease varies based on genetics and environmental factors, often with subclinical alterations in kidney function being overlooked. This study aimed to examine changes in kidney morphology before significant kidney function decline in the offspring of mice fed a low-protein diet. METHODS: Using a combination of histological analysis, kidney metabolic and hemodynamic panel assessments, and advanced statistical techniques such as Linear Discriminant Analysis (LDA) and Principal Component Analysis (PCA), we investigated the initial impact of a maternal low-protein diet (LPD) on kidney development and function. Our study utilized 12-week-old F1 mice from F0 parents fed either a low-protein diet (LPD) or a normal-protein diet (NPD) before the onset of hypertension. RESULTS: The offspring (F1 generation) of parents (F0 generation) fed an LPD show reduced body weight from birth to P20. The kidney weight was also reduced compared to F1 offspring from parents fed an NPD. At 12 weeks of age, body weight normalized, but kidney weight remained low. Offspring of parents fed an LPD displayed abnormal kidney morphology, including dilated tubules, oligonephropathy, and fluid-filled cysts which had worsened with age. A kidney metabolic panel analysis at 12 weeks revealed a slight but consistent increase in urine albumin, plasma creatinine, mean urea, and BUN concentrations. Although no significant changes in hemodynamic variables were observed, 2/12 mice, both males, showed alterations in systolic blood pressure, suggesting sex-specific effects when comparing F1 mice from F0 fed either diet. Overall, kidney metabolic changes were strongly correlated to parental LPD. CONCLUSION: Our findings indicate that significant kidney damage must accumulate in the F1 generation from parents fed an LPD before any detectable changes in blood pressure occur. Our study suggests that small variations in kidney metabolic function may point to early kidney damage and should not be overlooked in the offspring of these malnourished mice and likely humans.

Dual Effect of Carnosine on ROS Formation in Rat Cultured Cortical Astrocytes.

Carnosine is composed of ß-alanine and L-histidine and is considered to be an important neuroprotective agent with antioxidant, metal chelating, and antisenescence properties. However, children with serum carnosinase deficiency present increased circulating carnosine and severe neurological symptoms. We here investigated the in vitro effects of carnosine on redox and mitochondrial parameters in cultured cortical astrocytes from neonatal rats. Carnosine did not alter mitochondrial content or mitochondrial membrane potential. On the other hand, carnosine increased mitochondrial superoxide anion formation, levels of thiobarbituric acid reactive substances and oxidation of 2',7'-dichlorofluorescin diacetate (DCF-DA), indicating that carnosine per se acts as a pro-oxidant agent. Nonetheless, carnosine prevented DCF-DA oxidation induced by H2O2 in cultured cortical astrocytes. Since alterations on mitochondrial membrane potential are not likely to be involved in these effects of carnosine, the involvement of N-Methyl-D-aspartate (NMDA) receptors in the pro-oxidant actions of carnosine was investigated. MK-801, an antagonist of NMDA receptors, prevented DCF-DA oxidation induced by carnosine in cultured cortical astrocytes. Astrocyte reactivity induced by carnosine was also prevented by the coincubation with MK-801. The present study shows for the very first time the pro-oxidant effects of carnosine per se in astrocytes. The data raise awareness on the importance of a better understanding of the biological actions of carnosine, a nutraceutical otherwise widely reported as devoid of side effects.

The Impact of Low Protein Diet on the Molecular and Cellular Development of the Fetal Kidney.

Background: Low nephron number has a direct impact on the development of hypertension and chronic kidney disease later in life. While intrauterine growth restriction caused by maternal low protein diet (LPD) is thought to be a significant cause of reduced nephron endowment in impoverished communities, its influence on the cellular and molecular processes which drive nephron formation are poorly understood. Methods: We conducted a comprehensive characterization of the impact of LPD on kidney development using tomographic and confocal imaging to quantify changes in branching morphogenesis and the cellular and morphological features of nephrogenic niches across development. These analyses were paired with single-cell RNA sequencing to dissect the transcriptional changes that LPD imposes during renal development to affect nephron number. Results: Single cell analysis at E14.5 and P0 revealed differences in the expression of genes and pathways involved in metabolism, cell cycle, epigenetic regulators and reciprocal inductive signals in most cell types analyzed, yielding imbalances and shifts in cellular energy production and cellular trajectories. In the nephron progenitor cells, LPD impeded cellular commitment and differentiation towards pre-tubular and renal vesicle structures. Confocal microscopy revealed a reduction in the number of pre-tubular aggregates and proliferation in nephron progenitor cells. We also found changes in branching morphogenesis, with a reduction in cell proliferation in the ureteric tips as well as reduced tip and tip parent lengths by optical projection tomography which causes patterning defects. Conclusions: This unique profiling demonstrates how a fetal programming defect leads to low nephron endowment which is intricately linked to changes in both branching morphogenesis and the commitment of nephron progenitor cells. The commitment of progenitor cells is pivotal for nephron formation and is significantly influenced by nutritional factors, with a low protein diet driving alterations in this program which directly results in a reduced nephron endowment. Significance Statement: While a mother's diet can negatively impact the number of nephrons in the kidneys of her offspring, the root cellular and molecular drivers of these deficits have not been rigorously explored. In this study we use advanced imaging and gene expression analysis in mouse models to define how a maternal low protein diet, analogous to that of impoverished communities, results in reduced nephron endowment. We find that low protein diet has pleiotropic effects on metabolism and the normal developmental programs of gene expression. These profoundly impact the process of branching morphogenesis necessary to establish niches for nephron generation and change cell behaviors which regulate how and when nephron progenitor cells commit to differentiation.

Gene length is a pivotal feature to explain disparities in transcript capture between single transcriptome techniques.

The scale and capability of single-cell and single-nucleus RNA-sequencing technologies are rapidly growing, enabling key discoveries and large-scale cell mapping operations. However, studies directly comparing technical differences between single-cell and single-nucleus RNA sequencing are still lacking. Here, we compared three paired single-cell and single-nucleus transcriptomes from three different organs (Heart, Lung and Kidney). Differently from previous studies that focused on cell classification, we explored disparities in the transcriptome output of whole cells relative to the nucleus. We found that the major cell clusters could be recovered by either technique from matched samples, but at different proportions. In 2/3 datasets (kidney and lung) we detected clusters exclusively present with single-nucleus RNA sequencing. In all three organ groups, we found that genomic and gene structural characteristics such as gene length and exon content significantly differed between the two techniques. Genes recovered with the single-nucleus RNA sequencing technique had longer sequence lengths and larger exon counts, whereas single-cell RNA sequencing captured short genes at higher rates. Furthermore, we found that when compared to the whole host genome (mouse for kidney and lung datasets and human for the heart dataset), single transcriptomes obtained with either technique skewed from the expected proportions in several points: a) coding sequence length, b) transcript length and c) genomic span; and d) distribution of genes based on exons counts. Interestingly, the top-100 DEG between the two techniques returned distinctive GO terms. Hence, the type of single transcriptome technique used affected the outcome of downstream analysis. In summary, our data revealed both techniques present disparities in RNA capture. Moreover, the biased RNA capture affected the calculations of basic cellular parameters, raising pivotal points about the limitations and advantages of either single transcriptome techniques.

Acetyl-CoA is a key molecule for nephron progenitor cell pool maintenance.

Nephron endowment at birth impacts long-term renal and cardiovascular health, and it is contingent on the nephron progenitor cell (NPC) pool. Glycolysis modulation is essential for determining NPC fate, but the underlying mechanism is unclear. Combining RNA sequencing and quantitative proteomics we identify 267 genes commonly targeted by Wnt activation or glycolysis inhibition in NPCs. Several of the impacted pathways converge at Acetyl-CoA, a co-product of glucose metabolism. Notably, glycolysis inhibition downregulates key genes of the Mevalonate/cholesterol pathway and stimulates NPC differentiation. Sodium acetate supplementation rescues glycolysis inhibition effects and favors NPC maintenance without hindering nephrogenesis. Six2Cre-mediated removal of ATP-citrate lyase (Acly), an enzyme that converts citrate to acetyl-CoA, leads to NPC pool depletion, glomeruli count reduction, and increases Wnt4 expression at birth. Sodium acetate supplementation counters the effects of Acly deletion on cap-mesenchyme. Our findings show a pivotal role of acetyl-CoA metabolism in kidney development and uncover new avenues for manipulating nephrogenesis and preventing adult kidney disease.

Age-Associated Upregulation of Glutamate Transporters and Glutamine Synthetase in Senescent Astrocytes In Vitro and in the Mouse and Human Hippocampus.

Aging is marked by complex and progressive physiological changes, including in the glutamatergic system, that lead to a decline of brain function. Increased content of senescent cells in the brain, such as glial cells, has been reported to impact cognition both in animal models and human tissue during normal aging and in the context of neurodegenerative disease. Changes in the glutamatergic synaptic activity rely on the glutamate-glutamine cycle, in which astrocytes handle glutamate taken up from synapses and provide glutamine for neurons, thus maintaining excitatory neurotransmission. However, the mechanisms of glutamate homeostasis in brain aging are still poorly understood. Herein, we showed that mouse senescent astrocytes in vitro undergo upregulation of GLT-1, GLAST, and glutamine synthetase (GS), along with the increased enzymatic activity of GS and [3H]-D-aspartate uptake. Furthermore, we observed higher levels of GS and increased [3H]-D-aspartate uptake in the hippocampus of aged mice, although the activity of GS was similar between young and old mice. Analysis of a previously available RNAseq dataset of mice at different ages revealed upregulation of GLAST and GS mRNA levels in hippocampal astrocytes during aging. Corroborating these rodent data, we showed an increased number of GS + cells, and GS and GLT-1 levels/intensity in the hippocampus of elderly humans. Our data suggest that aged astrocytes undergo molecular and functional changes that control glutamate-glutamine homeostasis upon brain aging.

The Potential Use of Carnosine in Diabetes and Other Afflictions Reported in Long COVID Patients.

Carnosine is a dipeptide expressed in both the central nervous system and periphery. Several biological functions have been attributed to carnosine, including as an anti-inflammatory and antioxidant agent, and as a modulator of mitochondrial metabolism. Some of these mechanisms have been implicated in the pathophysiology of coronavirus disease-2019 (COVID-19). COVID-19 is caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2). The clinical manifestation and recovery time for COVID-19 are variable. Some patients are severely affected by SARS-CoV-2 infection and may experience respiratory failure, thromboembolic disease, neurological symptoms, kidney damage, acute pancreatitis, and even death. COVID-19 patients with comorbidities, including diabetes, are at higher risk of death. Mechanisms underlying the dysfunction of the afflicted organs in COVID-19 patients have been discussed, the most common being the so-called cytokine storm. Given the biological effects attributed to carnosine, adjuvant therapy with this dipeptide could be considered as supportive treatment in patients with either COVID-19 or long COVID.